The long-term objective of this research is a detailed understanding of herpesvirus DNA polymerases and drugs that target them. These enzymes, which include a catalytic subunit (Pol) and an accessory subunit that stimulates long-chain DNA synthesis, are both prototype ?-like DNA polymerases and excellent targets for antiviral drugs. This latter property is especially health-related, as new drugs are needed for treatment of herpesvirus infections. In this application, unanswered questions regarding accessory subunits, catalytic subunits, and drugs that target these proteins and their interaction are addressed. Specific aim 1 is to investigate the unusual and different manners by which the accessory subunits, such as herpes simplex virus (HSV) UL42 and human cytomegalovirus (HCMV) UL44, interact with DNA so that they bind tightly, yet diffuse linearly along the DNA to permit processive DNA synthesis. Single-molecule approaches will be used to analyze how these proteins move on DNA, particularly whether wild type UL42 or a tight-binding mutant necessarily moves helically or can, for example, move along one side of the helix. The force required to move these subunits will be compared with the force required to stop or slow the catalytic subunits. X-ray crystallography will be used to understand the molecular details of the protein-DNA interaction. Specific aim 2 is to investigate the roles of structural domains of the catalytic subunits that are N-terminal to the thumb, palm, and fingers domains in terms of enzymatic functions, binding to the base excision repair (BER) enzyme uracil DNA glycosylase (UNG), viral replication, and mechanisms of antiviral drug resistance. Two structural domains, pre-NH2 and NH2, have been observed in the crystal structure of HSV Pol, but their roles in enzyme function and viral replication are unknown. To address these questions, mutant enzymes will be engineered and assayed for relevant biochemical activities. Mutant viruses will be engineered and assayed for viral replication both in cells and in a mouse model. The mechanisms by which mutant HCMV Pols with substitutions in their 3'-5' exonuclease domain resist ganciclovir (GCV) action will be investigated using enzymological analyses. Specific aim 3 is to discover new compounds that inhibit the interaction of HCMV Pol and UL44, and HCMV replication, using a structure-based approach. The importance of the interaction between UL44 and another replication protein, UL84, for viral replication, and whether it can be exploited as a drug target, will be investigated using a combination of biochemical, molecular genetic, and cell biological approaches, including efforts to develop a new technique to map protein-protein interactions. Should the UL44-UL84 interaction look promising as a drug target, random screening for compounds that inhibit this interaction will be undertaken. PUBLIC HEALTH RELEVANCE: Herpesviruses cause widespread disease in the population at large and severe disease in people with impaired immunity. There is considerable need for new drugs to combat these viruses. The research proposed should not only provide information that could aid in drug discovery and understanding how viruses become resistant to current drugs, but aims directly to discover new anti-herpesvirus drugs.